This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. EGFR (the epidermal growth factor receptor) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands, being activated by binding to epidermal growth factor and transforming growth factor [unreadable] (TGF[unreadable]). Mutations that lead to EGFR overexpression have been associated with a number of cancers, including lung cancer and glioblastoma multiforme. To isolate EGFR with its interacting partners we tested several monoclonal and polyclonal commercially available anti-EGFR antibodies. However, none of these proved successful for immunoisolations. We therefore proceeded in testing in-house-developed antibodies (rabbit polyclonal), which we had to further purify to achieve antibodies usable for immunoisolations. After careful optimization of lysis buffers, and tests of magnetic beads versus agarose beads, we now have isolated EGFR with associated proteins. Our results confirmed previous known interacting partners, such as GRB2, and indicated numerous phosphorylation sites on EGFR. In our effort to obtain immunoisolations as clear as possible of background (non-specific binding), we tested the use of a filtration step prior to immunoisolations. Following filtration, we could still detect EGFR and GRB2. Having successfully isolated EGFR, we have now started the comparison between wild type and two mutant EGFR proteins.